Download Effects of Caffeine in Operant Learning and Locomotor Activity

Western University
Scholarship@Western
2015 Undergraduate Awards
The Undergraduate Awards
2015
Effects of Caffeine in Operant Learning and
Locomotor Activity
Daniel Kharlas
Western University, [email protected]
Follow this and additional works at: http://ir.lib.uwo.ca/ungradawards_2015
Part of the Psychology Commons
Recommended Citation
Kharlas, Daniel, "Effects of Caffeine in Operant Learning and Locomotor Activity" (2015). 2015 Undergraduate Awards. Paper 4.
http://ir.lib.uwo.ca/ungradawards_2015/4
Running head: CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
Effects of Caffeine in Operant Learning and Locomotor Activity
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CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Abstract
Caffeine is widely considered to be a reinforcer in humans, but this effect is difficult to measure
in non-human animals. This study extended the understanding of the effects of caffeine on
reinforcement and locomotor activity by testing rat animal models. A group of 24 adult male
Long Evans rats was split into three groups, caffeine (15mg/kg), Scopolamine (spatial learning
inhibitor) or a NaCl control. Each drug was administered 15 min prior to behavioural testing.
The rats were given one habituation session to familiarize with eating the reinforcing agent, one
baseline session before testing and on test day were monitored for the number of bar presses,
vertical movements, horizontal movement and latency to first reinforcement across six 2 minute
time bins. It was hypothesized that rats injected with caffeine would experience an increase in
motivation for food reward, presenting as a greater number of bar presses compared to the
control groups. Caffeine injected rats are expected to exhibit greater locomotor activity than
control groups. Rats injected with caffeine had greater locomotor movement but had no positive
reinforcement on bar presses and experienced a lower number of bar presses than the control
group. Thus, the results of this study suggest caffeine had a negative reinforcement effect on
operant learning mechanisms. Future studies should explore what could have caused this
negative reinforcement interaction between caffeine and learning of the bar pressing
reinforcement task.
Key words: Caffeine, Locomotor Activity, Reinforcement, Operant Learning, Motivation
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Introduction
Caffeine is currently the most commonly taken psychoactive drug, used by a large
percentage of the population mainly for its stimulant properties (Jarvis, 1993). Past studies have
solidified our understanding of caffeine as a stimulant. It has been cited to increase general
physiological arousal as measured by increased cortisol production, heart rate and respiration
(Fisone, Borgvist & Usiello, 2004). In addition to its stimulant properties, caffeine is often used
as a reinforcing agent in human studies (Griffths et al., 1988). While caffeine’s reinforcement
effects have been difficult to replicate in non-human subjects (Sheppard et al., 2012) its effects
on locomotor activity have been found in both human and non-human experiments.
Antoniou et al.’s 1998 study tested the effects of a number of drugs including caffeine,
amphetamines and cocaine on a number of paradigms to test for locomotor activity. The
researchers demonstrated that low and medium doses of caffeine produced higher locomotor
activity in rat subjects compared to control subjects who did not receive the drug. This discovery
is important for understanding how caffeine influences behaviour. To further understand the
mechanisms of caffeine use, recent studies have begun to explore how caffeine interacts with
other drugs.
In combination with cocaine, caffeine was found to increase excitation of subjects further
increasing locomotor activity; it also increased the reinforcement effects of cocaine (Mehta, Jain,
& Billie, 2004 and Kunin et al., 2000). Caffeine in combination with both amphetamines and
cocaine has been to shown significantly increase level of self-drug administration in both nonhuman primates and rats (Comer and Carroll, 1996). Current research has furthered the study of
caffeine on operant conditioning using reinforcing agents other than drugs. For example in 1993,
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Buffalo, Gillam, Allen and Paule, found that rhesus monkeys given caffeine showed behavioural
alterations that caused them to improve at both learning and reinforcement tasks.
According to the study conducted by Fernstrom and Fernstrom’s (1984) the mechanism,
of caffeine altering reinforcement, may be caused by changes in the release, reuptake and use of
dopamine in response to caffeine consumption. Dopamine is a neurotransmitter found in various
different brain regions, including the nucleus accumbens. It is a key player in the reward
pathways of the brain and in understanding the neural mechanism of why addiction and selfadministration of drugs, or non-drug reinforcers, work. The results of an investigation carried out
by Solina et al. in 2002 showed that caffeine facilitated dopaminergic neurotransmission by
stimulating dopamine release and potentiating dopamine receptor stimulation inside of the
nucleus accumbens. This resulted in greater activation of the reward pathway of the brain when
reinforcement in either drug or non-drug forms were used.
To understand how caffeine alters reinforcement and operant learning on a behavioural
level, researchers have measured operant learning using novel tasks. One example implemented
by Bergen, Spratt & Messier (1997) successfully measured operant learning using the bar
pressing task in the rat animal model. Other studies looking at the relationship between operant
learning and caffeine have focused on motivations for food reward in subjects (Sheppard et al.,
2012). These studies have not yet looked at how caffeine affects operant learning in food
reinforcement tasks. Sheppard et al.’s (2012) study demonstrated that caffeine indirectly
increased in the reinforcing effects of lever presses, leading to food reward, which in turn led to
more lever presses. Although this study clearly showed that caffeine causes increased motivation
for operate learning. The extent to which it acts as a reinforcer was not revealed.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Study by Smith, Hunter and Bennett (1994), demonstrated the use of Scopolamine as a
successful memory inhibitor in the Morris Water Maze task and therefore a successful control
group. It is expected that this effect would carry over to the bar pressing task in which rats must
first learn the connection between bar press and food reward. Scopolamine is expected to block
the learning of rats, decreasing performance levels in any bar-pressing food reward
reinforcement tasks. A reinforcement schedule using a bar pressing task and a food reward will
therefore be used to test operant learning of caffeine injected and control.
The current study plans to extend the study of caffeine and how it interacts with
locomotor activity of subjects by testing on medium doses of caffeine and exploring how
locomotor activity may change over time as caffeine is metabolized. Furthermore, this study
intends to explore the relationship of caffeine and reinforcement learning therefore extending the
understanding of how caffeine affects the rat animal model. It is hypothesized that rats injected
with caffeine will experience an increase in motivation for food reward, presenting as a greater
number of bar presses compared to the control groups. Caffeine injected rats are expected to
exhibit greater locomotor activity than control groups.
Method
Participants
Subjects were male Long Evans rats (325-375g) taken from Charles River, Quebec. The
animals were paired and housed in polypropylene cages in a colony room at 21 ± 1 ºC. Rats were
kept in a 12:12 light dark cycle that began at 0700h and habituated to a food deprivation
schedule while maintained at 90% of pre-deprivation body weight. Rats were handled, and tested
according to the guidelines that were set by the Canadian Council on Animal Care (CCAC).
Apparatus and Materials
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Subjects were placed in a operant testing chamber(43 cm X 35 cm X 30 cm) made up of
plywood with a single clear Plexiglas front panel with a retractable lever near a food pellet
dispenser that was designed to provide reinforcement for every bar press(FR-1). Locomotion was
measured by two observers and a chronometer was used to time latency to first response.
Drugs
Each of the subjects were injected with either 15 mg/kg caffeine (Lot# SLBH1002V;
Sigma, St. Louis, MO) dissolved in 0.9% saline, 1 mg/kg of scopolamine hydrobromide (Sigma,
St. Louis, MO) dissolved in 0.9% saline or a control injection of 0.9% saline. Volume of
injection was 1 ml/kg; all injections were given intraperitoneal (i.p.).
Procedure
Rats first received a habituation session in which they were placed inside the testing
chamber to familiarize themselves with eating the reinforcing agent (sweetened food pellets-Test
Diet purified rodent table 5TUL). One week before testing day rats received two 10 min shaping
sessions in the testing chamber, in which reinforcement tablets were given to cause rats to learn
that pressing the bar would release food. This was done by first reinforcing movements towards
the bar and eventually only reinforcing touching the bar until the rat eventually learned the press
the bar. At the end of the two 10 min sessions rats were expected to have learned that pressing
the bar would allow them to release food themselves. Three days before test day rats received
one baseline test session in which they were place in the testing chamber for 12 min and the
number of bar presses were monitored.
Rats were injected i.p. with one of the three drugs 15 minutes prior to placement in the
operant test chamber. Rats were assessed for 12 minutes split into 6 time bins. Locomotion was
assessed by looking at horizontal and vertical movements. A horizontal movement was defined
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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as any movement that resulted in two paws crossing any quadrant line in any direction. A
vertical movement was defined as any movement which resulted in the rats paws being off the
ground and its nose passing above the horizontal line inside the open-field apparatus. Number of
bar presses, rate of response and latency to first response were all measured during testing to
understand how the different drugs may affect operate learning of rats. A comparison between
pre-drug manipulation testing and post-drug manipulation testing of total number of bar presses
was also done. All instances of any movement, bar press or rate of response were recorded by
two observers and a third observer timed the latency to first response.
Treatment of Data
To test for inter reliability of both observers for horizontal and vertical movement a
Pearson correlation test was used. The correlation was considered significant if r > 0.5.
Horizontal movement, vertical movement and response rate were analyzed using a 3 x 6 splitplot ANOVA. The ANOVA was done between the different groups at 3 levels (caffeine,
scopolamine and NaCl control) within 2 minute time bins at 6 levels. Latency for first response
was analyzed by comparing the average of each between subjects group (caffeine, scopolamine
and NaCl control) using a between subjects one way ANOVA. A 3 x 2 split-plot ANOVA was
used for comparing the total number of responses for pre and post drug manipulations between
groups at 3 levels (caffeine, scopolamine and NaCl control) and within 2 levels of total responses
(pre and post drug manipulation). A criterion of p < .05 was used for all ANOVA tests.
Results
Horizontal Movement
A high inter-rater reliability factor was found between observers, therefore justifying the
use of only one set of data for analysis, r (21) = 0.94. The results of the 3 x 6 split-plot ANOVA
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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showed a significant main effect of drug group on horizontal movement (F (2, 21) = 48.50, p <
.001). Specifically, a significant different was observed between Caffeine and Scopolamine
drug groups for horizontal movement. A significant difference between Caffeine and the control
NaCl group was also observed for horizontal movement. Scopolamine and the control NaCl
group did not significantly differ for horizontal movements (see Figure 1). A significant main
effect of time bins on horizontal movement was found (F (5, 21) = 16.59, p < .001). Generally a
significant decrease was seen across time bins (see Figure 1). A significant interaction effect
between drug group and time bins was found, (F (10, 105) = 2.85 p < .01). Both Caffeine and
control NaCl groups showed a decrease over time bins while, Scopolamine showed very little
change over the six time bins. Over six time bins, Caffeine and NaCl control groups experienced
a large decrease in horizontal movements while Scopolamine stayed consistent. Caffeine
experienced the greatest decrease in horizontal movement across the 6 time bins and also
demonstrated the greatest number of horizontal movements.
Vertical Movement
A high inter-rater reliability factor was found between observers, therefore justifying the
use of only one set of data for analysis, r (21) = 0.90. The results of the 3 x 6 split-plot ANOVA
showed a significant main effect of drug group on vertical movement (F (2, 21) = 5.53, p <
.001). The main effect of drug groups was likely driven by the significantly greater vertical
movement found in the caffeine group in comparison to Scopolamine and control groups (see
Figure 2). A significant main effect of time bins on vertical movements was found (F (5, 21) =
16.49, p < .001). The significant main effect of time bins was likely driven by the decrease in
vertical movements across the 6 time bins (See figure 2). A significant interaction effect was
found for drug groups within time bins (F (10, 105) = 3.18, p < .01). Specifically, caffeine and
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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NaCl control groups both showed a significant decrease in vertical movement in the first time
bin which than leveled out into a steady number of vertical movements, while Scopolamine
showed a non-significant change in vertical movement across the 6 time bins(see Figure 2).
Across all 6 time bins Caffeine was observed to significantly decrease while Scopolamine
showed no change in vertical movement. The Caffeine group demonstrated the greatest number
of vertical movements throughout most of the observation period.
Response Rate
The data analysis using a 3 x 6 split-plot ANOVA revealed a significant main effect of
drug group on response rate (F (2, 21) = 93.84, p < .001). The Caffeine group showed a
significantly lower response rate when compared to the control NaCl. The Caffeine group also
showed a significant greater response rate than the Scopolamine group (See Figure 3). A
significant main effect of time bins on response rate was found (F (5, 21) = 16.49, p < .001). An
increase from time bins 1 to 2 followed by a steady decrease of response rate from time bins 2-5
and a flattening out from time bins 5-6 was the general trend found in figure 3. A significant
interaction effect was found for drug groups within time bins for response rates (F (10, 105) =
3.29, p < .01). Scopolamine injected rats had no significant change from time bins 1-2 followed
by a decrease from time bins 2-3 than a flattening out with no change from time bins 3-6 the
trend of caffeine and NaCl control group within time bins was significantly different.
Specifically, Caffeine and NaCl control groups declined significantly from time bins 2-5 while
the Scopolamine group showed no significant change during this time period. In summary, the
greatest response rate was found in control NaCl groups. The Caffeine group demonstrated a
significantly lower response rate than the control group. Both the control and caffeine groups
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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showed an increase of response rate at the beginning of the experiment followed by a steady
decrease in response rate until the 6th time bin.
Latency to first response
A one way between subjects ANOVA demonstrated a significant difference between
drug groups for latency to first response (F (2, 21) = 5.07, p = .02). Caffeine and control group
did not significantly differ for latency to first response. Both Caffeine and control groups
demonstrated significantly shorter latencies to first response when compared to the scopolamine
drug group (see Figure 4).
Total number of pre vs. post drug manipulation responses
The 3 x 2 split-plot ANOVA analysis revealed a significant main effect of drug
manipulation on total number of responses(F(2, 21) = 50.85, p < .001). Specifically, the
Scopolamine group was observed to have a significantly different number of responses than
either the control NaCl or Caffeine group. The caffeine group and Control groups did not
significantly differ on their total number of responses(see Figure 5) The pre drug manipulation
had a significantly greater number of total responses than the post drug manipulation (F(1, 2) =
156.93, p < .001). A significant interaction between drug group within pre and post drug
manipulation was observed (F(2, 21) =61.38, p < .001). To be exact, the Scopolamine group
significantly differed in pre and post drug manipulation for total number of responses while,
Caffeine and control groups did not differ significantly between pre and post drug
manipulations(see Figure 5). The Scopolamine drug group demonstrated an effect of drug
manipulation on total number of responses for pre and post drug manipulation. In post drug
manipulations Scopolamine demonstrated a significantly lower number of responses while the
Caffeine group showed no significant difference.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Discussion
It was hypothesized that caffeine injected rats were expected to experience greater
locomotor activity than control groups. Rats injected with caffeine were also expected to
experience an increase in motivation for food reward and therefore display a greater number of
bar presses than the control groups.
The locomotor activity of caffeine-injected rats supported the hypothesis of this study.
The caffeine group was found to have a greater number of both vertical and horizontal
movements throughout the experiment than either the control or the Scopolamine group. This
adds support to previous findings that specific doses of caffeine increase locomotor activity
(Bedingfield, King and Holloway, 1998). It is likely that a dose greater than the 15mg/kg used in
this study may lead to a decrease in locomotor activity. In Antoniou et al.’s 1998 study it was
found that a dose of 20 mg/kg would have an opposite effect to the group given 5 or 10 mg/kg of
caffeine. These results are contrary to results found in 1982 by Carney in which 20 mg/kg of
caffeine was found to be a peak concentration for locomotor activity.
When comparing the amount of reinforcement bar presses between the pre-drug
manipulation data and the post-drug manipulation data no significant difference was detected for
the caffeine group. This is supported by results of latency to first bar press which did not differ
significantly between the caffeine and control groups. This likely means caffeine did not help the
subject to learn the relationship between bar press and reward any faster than the saline control.
Furthermore, the control group was found to have significantly more bar presses per time bin
than the caffeine group. It is possible that caffeine had a negative effect on reinforcement of food
reward. This indicates that the results do not support the hypothesis, and that caffeine did not
have a positive effect on reinforcement over the control and Scopolamine injected group.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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The negative reinforcement of bar presses observed with the caffeine group may have
been caused by a number of factors. Two human caffeine ingestion studies (Jessen et al., 2004
and Belza, Toubro & Astrup, 2009) found that caffeine itself was able to suppress appetite and
that it could increase appetite suppression caused by other drugs. This lowered appetite would
lead to decreases in the need for food reinforcement. This would imply that the experiment is no
longer viable for measurement of operant learning, since the reward is no longer desirable
enough to the subject to create a reliable conditioned stimulus reaction.
Another theory of why caffeine may have caused negative reinforcement is the dosage
used in the current study. A dose of 15mg/kg may have been too high, leading rats to lose
motivation to seek food rewards. This occurred in Buffalo et al.’s, (1993) study in which rhesus
monkeys injected with higher doses of caffeine showed less conditioned responses to the
conditioned stimulus due to lack of motivation leading to a learning deficit. This lack of
motivation may be become present in rats injected with 15mg/kg of caffeine as well. To solidify
which theory is driving the effect or if both begin to play a part at 15 mg/kg further studies
should replicate this study while replacing control groups with various doses of caffeine. These
future studies would make it clear whether doses of caffeine may play a part in how caffeine
alters operant learning, motivation and general reinforcement of non-drugs.
The current study looked mainly at the effects of caffeine on operant learning in
comparison to Scopolamine and a saline control group as well as the difference in locomotor
movement when subjects were injected 15 minutes prior to the experiment. However future
studies should study how different schedules of administration may change its effects on
learning. Previous findings in studies observing the effects of caffeine on spatial learning
paradigms have indeed found variation in results when testing different schedules of
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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administration (Angelucci, Cesario, Hiroi, Rosalen & Cunha, 2002). This warrants future
research into whether variations in schedules of administration of caffeine strengthen or weaken
the reinforcing effect of non-drug rewards. Further applying principles in this study to human
studies would also allow for greater generalization of results, namely the effects of caffeine on
conditioned responses to non-drugs. Further studies are warranted to test the theories of negative
reinforcement caused by decreases in motivation. These studies should attempt to replicate the
current study while using different non-drug reward stimuli that are not food. To further
understand how caffeine may be an appetite suppressor more research should be conducted
specifically looking at caffeine’s effects on appetite. Future studies should also explore which
dose of caffeine produces the peak locomotor activity and which doses would begin to lead to a
decrease in locomotor activity. This research can then be used to create a better procedure for
testing caffeine’s effects on operant learning.
In conclusion, the first hypothesis of this study was supported. Caffeine caused increases
in locomotor activity as was demonstrated by significant increases of horizontal and vertical
movement over both control groups. The second hypothesis was not supported in this study. In
fact, caffeine was found to be a negative reinforcer of the bar pressing task and demonstrated a
significantly smaller number of bar presses in compared to the NaCl control group. This may
have been caused by caffeine causing a lack of motivation or suppressing appetite. Further
research needs to be done on both theories so as to understand whether food as a reward is viable
or may be causing a negative reinforcement. This study adds to the body of literature stating that
caffeine effects locomotor activity. The results of the current study warrants more research into
how caffeine interacts with operant learning.
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CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Horizontal Movements
35
# of Horizontal movements
30
Scopolamine
25
NaCl
20
Caffeine
15
10
5
0
1
2
3
4
5
6
Time bins (2 minutes each)
Figure 1. The mean number of horizontal movements of subjects (n=24) across 6 two minutes
time bins. Mean horizontal movements ± SE of male Long Evans rats observed 15 minutes after
intraperitoneal injection of caffeine (15mg/kg), NaCl control or scopolamine (1mg/kg) as a
function of six, 2 minute time bins. The caffeine group had a significantly greater number of
horizontal movement than either control or scopolamine groups. A significant difference was
observed at the first time bin, in general a decrease was seen across time bins. An interaction
effect between caffeine and scopolamine within the 6 time bins was observed. Caffeine and NaCl
both demonstrated a significant decrease in horizontal movements across 6 time bins. Error bars
represent standard error of means.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Vertical Movements
8
Scopolamine
7
NaCl
Caffeine
# of vertical movements
6
5
4
3
2
1
0
1
2
3
4
5
6
Time bins (2 minutes each)
Figure 2. Vertical Movement across six time bins for each subject(n=24). Mean vertical
movements ± SE of male Long Evans rats observed 15 minutes after intraperitoneal injection of
caffeine (15mg/kg), NaCl control or scopolamine (1mg/kg) as a function of six, 2 minute time
bins. A significant main effect of drug group was likely driven by the significantly greater
number of vertical movements in the caffeine group compared to the Scopolomine and control
groups. Both NaCl and Caffeine groups significantly decreased across time bins. While across
all 6 time bins Scopolamine had a non-significant change in the number of vertical movements.
Error bars represent standard error of means.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Rate of responses on test day
30
Scopolamine
NaCl
# of bar press responses
25
Caffeine
20
15
10
5
0
1
2
3
4
5
6
Time bins (2 minutes each)
Figure 3. The response rate after drug manipulation across six 2 minute time bins for subjects
(n=24). The mean number of bar presses ± SE of male Long Evans rats observed 15 minutes
after intraperitoneal injection of caffeine (15mg/kg), NaCl control or scopolamine (1mg/kg) as a
function of six, 2 minute time bins. The saline group drove the main effect of drug group as it
was found to have a significantly greater response rate than both caffeine and Scopolamine
groups. The control and Caffeine groups both started at a high number of test day responses and
experienced an increase in number of test day of responses per bin that was followed by a
decrease until the end of the experiment this was significantly greater than the Scopolamine
group which had a low amount of test day responses per bin, throughout the time bins with no
significant change within time bins. Error bars represent standard error of means.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
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Latency to first response
120
100
Time (Seconds)
80
60
40
20
0
Scopolamine
NaCl
Caffeine
Drug Groups
Figure 4. Latency to first response between three drug groups for subjects(n=24). Scopolamine,
NaCl and Caffeine groups were injected intraperitoneal 15 minutes prior to operate behavior
observation testing bar presses which lead to food reward. Latency to first bar press response was
measured in seconds using a chronograph and compared between Scopolamine, NaCl and
caffeine drug groups. The Scopolamine injected group showed a significantly longer latency to
first response than Caffeine and NaCl drug groups. Caffeine and NaCl drug groups did not
significantly differ for the latency time to first bar press. Error bars represent standard error of
means.
CAFFEINE IN OPERANT LEARNING AND LOCOMOTOR ACTIVITY
140
20
Total number of responses pre and post drug
manipulation
Total # of responses
120
100
80
60
Pre-drug manipulation
40
Post-drug manipulation
20
0
Scopolamine
NaCl
Caffeine
Drug Groups
Figure 5. The total number of responses for pre-drug manipulation plotted against the total
number of responses after a drug manipulation ± SE of male Long Evans rats observed 15
minutes after intraperitoneal injection of caffeine (15mg/kg), NaCl control or scopolamine
(1mg/kg). The Scopolamine group showed a significant decrease for the total number of
responses from pre to post drug manipulation. The total number of responses for post-drug
manipulation for the Scopolamine group was significantly lower than either NaCl or Caffeine
groups. There was no significant difference between the three drug groups for pre-drug
manipulation. No significant difference was observed between NaCl and Caffeine groups for the
total number of response for post-drug responses. No significant difference was shown for the
caffeine group from pre-drug manipulation to post-drug manipulation for total number of
responses. Error bars represent standard error of means.